Figure 3.
Confirmation of TyrRS∗/tRNACUA as the second orthogonal pair
(A) (i) The reporter to test efficiency of TyrRS∗/tRNACUA. (ii) Chemical structure of OMeY. (iii) Chemical structure of AzF. (iv) OMeY or AzF incorporation efficiency by TyrRS∗/tRNACUA in HEK293 cells by using flow cytometry. Higher eGFP fluorescence was detected with AzF supplementation.
(B) Orthogonality of tRNA/aaRS pairs. HEK293 cells transfected with PylRS/tRNAUCCU(Ev2) or TyrRS∗/tRNACUA reporter vector and incubated for 24 h in the presence of AzF or BocK respectively. Fluorescent eGFP was only detected when the aaRS/tRNA pairs were in the presence of their cognate unnatural amino acid depicting mutual orthogonality of the pairs.
(C) Reporters for testing the impact of tRNA copy number on unnatural amino acid incorporation efficiency.
(D) Comparison of Pyl tRNAUCCU(Ev2) n = 1 and n = 4.
(E) Comparison of Tyr tRNACUA n = 1 and n = 4. Flow cytometry was used to analyze BocK or AzF incorporation into the 150th residue of eGFP. Unnatural amino acids were supplemented into the cell growth medium to a final concentration of 1 mM. Means and standard deviations calculated from three biological replicates are shown. For incorporation efficiency calculation, see Figure 2.